1. Field of the Invention
The present invention relates to methods and kits for the detection mutations associated with thrombosis.
2. Description of the Prior Art
Venous thromboembolism (VTE) disease also called venous thrombosis, which manifests clinically as deep vein thrombosis (DVT) and pulmonary embolisms (PE), represents a major health problem worldwide. A DVT is a blood clot that forms in leg veins. A PE is caused by a blood clot, typically in the leg, groin, or pelvic veins (and occasionally in upper extremity veins), which breaks free and travels to the lung arteries. A DVT is often the source of a clot that travels to the lung arteries and becomes a PE.
VTE is a multifactorial disease which is caused not only by multiple genetic factors but also by multiple acquired or environmental risk factors, such as surgery, use of oral contraceptives, hormone replacement therapy, and advanced age. The incidence of symptomatic venous thrombosis cases is approximately 1 in 1000 people per year. Various gene-gene, gene-environment, and environment-environment interactions between risk factors work synergistically to increase the risk of an individual to VTE. Despite recognition of risk factors and availability of pharmacologically effective options for prophylaxis, DVT and PE remain common causes of morbidity and mortality.
Several single nucleotide polymorphisms (SNPs) associated with VTE have been identified. The factor V Leiden polymorphism (G1691A) (Genbank Accession #Z99572) has been identified as the most common inherited cause, and is implicated in 20 to 40% of venous thrombosis cases. Heterozygotes possess a 3 to 7-fold increased risk of thrombosis while homozygous mutants carry a 50 to 100-fold increased risk. The second most common cause of inherited thrombophilia involves the factor II (prothrombin) G20210A polymorphism (Genbank Accession #M17262). This mutation results in elevated levels of factor II through mRNA stabilization and accounts for 6 to 8% of venous thrombosis cases. Factor II heterozygotes carry about a 2 to 5-fold increased risk of venous thromboembolism in the absence of other risk factors and a more than additive synergistic risk when other risk factors, especially oral contraceptive use, are present. Approximately 10 to 12% of factor V heterozygotes with venous thrombosis also carry the factor II mutation. A third independent risk factor for thrombosis, is the methylenetetrahydrofolate reductase (MTHFR)C677T polymorphism (Genbank Accession #NM005957.1). This mutation produces an MTHFR protein with reduced activity resulting in an increased serum level of homocysteine. This polymorphism is extremely common with homozygote frequencies of 5 to 15%. Furthermore, this polymorphism is also a risk factor for atherosclerotic heart disease, pre-eclampsia and fetal neural tube defects. There is a general consensus that the three aforementioned SNPs are the most important mutations associated with thrombophilia. Three additional SNPs, MTHFR A1298C (Genbank Accession #NM005957.1), factor XIII val34leu (factor XIII G4377T Genbank Accession #AF418272) and tissue factor plasma inhibitor (TFPI) C536T (GenBank Accession #M59497), are believed to have little or no independent effect on venous thrombosis. However, they may act synergistically with other genetic or acquired risk factors resulting in a more than additive effect or, in the case of factor XIII val34leu, a protective effect.
Individual polymorphisms, as described above, may have little or no independent effect on venous thrombosis but may act synergistically with other genetic or acquired risk factors, resulting in a more than additive effect. While approaches for providing this information by analyzing one gene at a time are currently available, the ability to detect all of the above mutations simultaneously would be extremely useful in establishing a complete picture of a patient's genetic risk profile.
Methodologies which can be used to detect the above mentioned SNPs are characterized by specific deficiencies. There are currently no rapid methods for determining several mutations across a number of genes simultaneously. For example, DNA sequencing of the above mentioned alleles for a large number of samples would require several days for simplex Polymerase Chain Reaction (PCR) amplification of individual SNPs, amplicon purification, sequencing reaction set-up and electrophoresis. This would have to be repeated for each of the amplicons generated representing the individual SNPs. Analysis of the sequencing data involves additional time.
Multiplex Allele Specific Primer Extension and Solid Support Detection of SNPs
Multiplex allele specific primer extension, and hybridization of extended primers to a solid support is described generally in the prior art. ASPE technology has been generally described in U.S. Pat. No. 4,851,331. The technology is designed to identify the presence or absence of specific polymorphic sites in the genome.
Multiplex ASPE in conjunction with hybridization to a support for mutation detection can be described generally as follows:
1) Amplifying regions of DNA comprising polymorphic loci utilizing a multiplexed, PCR.
2) Allele specific extension of primers wherein the amplified regions of DNA serve as target sequences for the allele specific extension. Extension primers that possess a 3′ terminal nucleotide which form a perfect match with the target sequence are extended to form extension products. Modified nucleotides are incorporated into the extension product, such nucleotides effectively labelling the extension products for detection purposes. Alternatively, an extension primer may instead comprise a 3′ terminal nucleotide which forms a mismatch with the target sequence. In this instance, primer extension does not occur unless the polymerase used for extension possesses exonuclease activity.
3) Hybridizing the extension product to a probe on a solid support, such as a microarray, wherein the probe is complementary to the 5′ end of the extension product.
The extension primers used in a methodology as described above, possess unique sequence tags at their 5′ ends. For example, the sequence tags may allow the extension products to be captured on a solid support.
Variations of the above technology have been described, for example, in U.S. Pat. No. 6,287,778 and PCT Application (WO 00/47766).
It is an object of the present invention to provide a method for the detection of variants associated with thrombosis.